Summary Interleukin-4 is a prototypic immunoregulatory cytokine. It is the central regulator of allergic inflammatory responses, controlling the polarization of naive CD4 T cells to the Th2 phenotype and Ig class switching to IgE. The Cytokine Biology Unit has characterized the signaling mechanisms utilized by the IL-4 receptor. It has shown that activation of the latent transcription factor, Stat 6, controls both TH2 polarization and IgE class switching and that GATA3 and Stat5 play essential roles in the acquisition of IL-4 producing capacity. Unit scientists have carried out genome-wide analyses of histone modification and transcription factor binding in each of the various Th lineages to gain insight into the processes underlying Th differentiation. They have identified a large number of sites to which GATA3 binds and sites for other transcription factors that bind in close proximity suggesting that these transcription factors act together with GATA3 in mediating gene activation and expression. This information has provided important insights into the genetic regulation of Th2 differentiation. Our analysis has shown that GATA3 mediates specific repressive functions in lineages other than Th2 cells and thus contributes to shaping the pattern of gene expression in various cell populations. Unit scientists have shown that nave CD4 T cells respond to T cell receptor (TCR) mediated weak signals by activating IL-4 transcription. This early TCR-dependent IL-4 production constitutes the induction phase of Th2 differentiation, followed by an IL-4-dependent polarization phase that completes Th2 differentiation. Both GATA3 and STAT5 were shown to be critical for this TCR-mediated early IL-4 transcription. It has now been demonstrated that such early TCR-dependent IL-4 production also critically requires two E proteins, HEB and E2a, which form a heterodimeric transcription factor. The reliance on weak TCR-mediated stimulation has been demonstrated to be due to T cell receptor-mediated activation of the ERK signaling pathway that blocks Th2 differentiation by preventing transcription of GATA3 and desensitizing the IL-2 receptor preventing activation of STAT5. Strong TCR-mediated signals stimulate ERK phosphorylation, thereby preventing TH2 differentiation and accounting for poor Th2 differentiation at high antigen concentration. Low concentrations of antigen, which activate ERK only weakly, are permissive for early IL-4 production and TH2 differentiation. The targets of ERK action that mediate suppression of GATA3 transcription are under active study as are the transcription factors that mediate TCR induced GATA3 transcription. We have confirmed that Notch signaling participates in Th2 priming mainly at low TCR signal strength and have shown that it does so by regulating both early IL-2 production and the sensitivity of the IL-4 receptor to IL-4 by increasing the level Stat-6 phosphorylation to a standard challenge of IL-4. The latter is due to repression of PTP1B expression by Notch signaling. Knocking down PTP1b expression in CD4 T cells enhances their Th2 differentiation in the absence of Notch. In addition, the defect in Th2 priming in mice with defects in the Notch pathway can be rescued by the addition of IL-2. Thus repressing PTP1b and enhancing IL-2 largely rescue the Notch defect in Th2 priming. In the course of studying control of Th2 differentiation by Notch, the discovery was made that deletion of presinilin (PS), a key component of gamma-secretase, which is essential for Notch function, leads to an unexpected result. The PS-deficient cells can efficiently differentiate into IL-4-producers when stimulated strongly through the TCR. They recover from ERK-mediated inhibition of signaling through the IL-2 receptor/ Stat5 pathway substantially more quickly than wild-type cells, suggesting that PS may work in parallel with ERK in desensitizing the IL-2 receptor. PS is a componmenet of the gamma secretase enzyme complex, which plays a central role in Notch signaling by cleaving Notch to generate the Notch intracellular domain. Since Pofut knockout cells, which fail to express a functional Notch, do not display the same phenotype as the PS-deficient cells, it does not appear that PS deficiency functions through the Notch pathway to allow high dose induction of Th2 cells. However, it seems clear that gamma-secretase is the critical enzyme since deficiency in a second component of the gamma secretase complex, nicastrin, leads to a phenotype similar to that seen with a PS deficiency. The enzyme substrate that gamma secretase acts on to prevent high antigen dose induction of an IL-4 response is now being sought. LI scientists have also developed new insights into the cytokine-stimulated production of IL-13 by basophils and mast cells and of IL-4 production by eosinophils. IL-3 activating STAT5 collaborates with IL-33 to activate mast cells IL-13 production; by contrast basophil IL-13 production can be stimulated most efficiently by IL-3 plus IL-18. Eosinophils are a rich source of IL-4, although much controversy still exists as to its mode of production and to the inducing ligands. Very recently, members of the Cytokine Biology Unit have clarified the complexity of the population of innate lymphoid cells (ILCs) that produce type II cytokines. They have shown the existence of a previously poorly appreciated cell population, inflammatory ILC2 (iILC2) cells that only appear in measureable numbers after the natural or artificial induction of IL-25. iILC2 cells express IL-25 receptors but not IL-33 receptors. However, both in vivo and in vitro, after the initial massive expansion in response to IL-25, they lose the expression of the IL-25 receptor and turn on IL-33 receptors and thereafter behave similarly to conventional ILC2 cells, particularly in response to helminth infection. Indeed, these cells play a major role in protection against N. brasiliensis infection. However, under different conditions of infection or to different inductive conditions, they can develop into ILC3-like cells and provide partial protection to Candida albicans infection. The iILC2 cells appear to be a unique cell population that provides a massive response/differentiation in cases of appropriate stimulation. Laboratory scientists have developed a series of indicator mice. Particularly valuable are those that reflect the expression of IL-4, IL-13 and TSLP. These indicator mice were made through the introduction of a bacterial artificial chromosomes in which the AM-Cyan gene reported IL-4, destabilized DS-Red has reported IL-13 and ZS-green reported TSLP expression. These mice have proved to be excellent reporters of cytokine production in Schistosomiasis infection and their value in other infections is being studied.